Cloning PCR products

One factor to watch out for is U.V. exposure. A large plasmid such as
yours makes a larger target for U.V. damage than do the 2-3 kb
varieties. Be sure to block as much U.V. as possible when excising from
the gel, allowing just enough fluorescence to see your DNA, and excise
it as quickly as possible. I found that our light box was able to
destroy all of a 3 kb plasmid after about 5-6 minutes.
As a control to see if this is a problem, just try re-ligating some cut
plasmid, without insert, before and after gel-purification.
Geoffrey Kidd
Matthew Knight wrote:
>> Hi all,
>> I am currently trying to clone a 0.65 PCR prod into a 7.2kb vector
> with great difficulty. I am using two different enzymes and have tried
> several different ligation ratios of 1:1, 3 insert: 1 vector and 5 insert
> : 1 vector.
>> I am digesting my PCR product and I am sure it is cutting due to a
> size differnece on agarose gel and then I am heat inactivating and ethanol
> precipitating the DNA.
>> I am gel purifying my vector with a silica matrix. My control
> transformation is working fine into E.coli TG2.
>> Does anyone have any suggestions on ligation ratios and DNA
> concentrations. For example I am ligating approximately 30-50ng vector
> with between 5-15ng insert.
>> Thanks in advance.
>> Cheers
> Matthew Knight
> Centre for Bioprocessing and Food Technology
> Victoria University
> Phone 61 3 9216 8137
> Fax 61 3 9216 8135